Manipulation of negative stranded RNA viruses by rearrangement of their genes and uses thereof

ABSTRACT

The present invention provides a method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, and a recombinant virus constructed by such method. Also provided is a method of attenuating a virus of the order Mononegavirales, and of constructing an attenuated virus useful for a vaccine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part and claims the benefit ofpriority under 35 USC §120 of U.S. patent application Ser. No.09/071,606, filed May 1, 1998, now U.S. Pat. No. 6,136,585, which claimsbenefit of priority under 35 U.S.C. 119(e) of U.S. provisionalapplication Ser. No. 60/045,471, filed May 2, 1997, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularvirology and vaccinology. More specifically, the present inventionrelates to the attenuation of negative stranded RNA viruses byrearrangement of their genes and uses thereof.

2. Description of the Related Art

The order Mononegavirales is composed of four families, theRhabdoviridae, the Paramyxoviridae, the Filoviridae and theBornaviridae. The viruses in these families contain a single strand ofnon-segmented negative-sense RNA and are responsible for a wide range ofsignificant diseases in fish, plants, and animals (Wagner, 1996). Theexpression of the genes encoded by these viruses is controlled at thelevel of transcription by the order of the genes on the genome relativeto the single 3′ promoter. Gene order throughout the Mononegavirales ishighly conserved: genes encoding products required in stoichiometricamounts for replication are always at or near the 3′ end of the genomewhile those whose products are needed in catalytic amounts are morepromoter distal (Pringle and Easton, 1997).

Vesicular stomatitis virus (VSV) is the prototypic virus of theRhabdoviridae. Its 11 kilobase genome has 5 genes which encode the 5structural proteins of the virus; the nucleocapsid protein, N, which isrequired in stoichiometric amounts for encapsidation of the replicatedRNA; the phosphoprotein, P, which is a cofactor of the RNA-dependent RNApolymerase, L; the matrix protein, M; and the attachment glycoprotein,G. The order of genes in the genome is 3′-N-P-M-G-L-5′ and previousstudies have shown that expression is obligatorily sequential from asingle 3′ promoter (Ball and White, 1976). Due to attenuation at eachgene junction the 3′-most genes are transcribed more abundantly thanthose that are more promoter distal (Iverson and Rose, 1981).

In nature, VSV infects a wide range of animals of which horses, cattle,and domestic swine are the most economically important. Infectionresults in the appearance of lesions around the mouth, hooves, and udderteats and while seldom fatal it leads to a loss in meat and milkproduction along with the expense of quarantine and vaccination. Thereare two main VSV serotypes, Indiana (Ind) and New Jersey (NJ) and whilethese viruses are endemic in Central and South American countries,outbreaks do occur within the United States. A recent outbreak in theU.S. occurred in 1997 in horses, and was of the Ind serotype whileprevious cases identified in 1995 and 1982-1983 were of the NJ serotype.The ease with which these viruses are transmitted, and the similarity oftheir symptoms to those caused by foot-and-mouth disease virus in cattleand domestic swine, makes VSV a pathogen of concern to the agricultureindustry.

Live attenuated viruses capable of replicating to generate protectivehumoral as well as cell mediated immune responses without producingdisease manifestations have proven effective vaccines against virusessuch as smallpox, yellow fever and poliomyelitis. The strategy forattenuation, however, has been empirical in most cases and notreproducible for general use. An additional consideration in the case ofRNA viruses is that the high error rate of RNA dependent RNApolymerases, their lack of proof reading and the quasi-species nature ofRNA virus populations (Domingo et al, 1996), make the use of liveattenuated viruses for this large group of medically significantpathogens problematic. This is especially true if the vaccine virus isbased on a limited number of single base changes as reversion tovirulence is a potential problem. For example, only a few back mutationscan restore virulence to the Sabin poliovirus type 3 vaccine strain(Wimmer et al., 1993).

The non-segmented negative strand RNA viruses of the familyMononegavirales possess an elegantly simple means of controlling theexpression of their genes. The linear, single-stranded RNA genomes ofthis family encode five to ten genes, the order of which is highlyconserved among all members. The prototype virus of this family is theRhabdovirus, vesicular stomatitis virus (VSV). Transcription of theviral genome is carried out by the virus-encoded RNA dependent RNApolymerase. There is a single entry site on the linear genome for theRNA polymerase, yet the mRNAs of the virus are not produced in equimolaramounts.

Available evidence indicates that the linear order of the genes on thegenome controls the levels of expression of individual genes.Transcription initiates at the single polymerase entry site at the 3′terminus of the genome and is obligatorily processive (Ball and White,1976). The level of expression of the individual genes a s monocistronicmRNAs is controlled by the dissociation, approximately 30% of the time,of the polymerase at each intergenic junction, as it traverses thegenome in the 3′ to 5′ direction (Iverson and Rose, 1981). Thismechanism of transcription results in sequentially decreasing amounts ofthe transcripts of each gene as a function of the distance of the genefrom the 3′ terminus of the genome. Correspondingly, gene productsneeded in stoichiometric amounts to support replication, such as thenucleocapsid (N) protein, are encoded at or near the 3′ terminus in allcases and expressed in the highest molar amounts (Villarreal et al.,1976, Ball and White, 1976). Gene products needed in enzymatic amounts,such as the RNA polymerase are encoded most distal from the 3′ end. Inall of the Mononegavirales, the polymerase gene is the 5′-most gene, andit is expressed in the lowest amount. Precise molar ratios of theproteins are required for optimal replication. For successfulreplication, proteins must be expressed in molar ratios that approximatethose expressed normally from the genome (Pattnaik and Wertz, 1990).

Viruses of the family Mononegavirales do not undergo homologous geneticrecombination (Pringle, 1987). Thus, other than defective interferingparticles, which lack portions of the genome, variants of these viruseshaving the entire complement of genes in a rearranged format have notbeen observed in nature.

The prior art is deficient in the lack of effective means of increasingexpression of a promoter distal gene in a virus of the orderMononegavirales and uses of such viruses. The present invention fulfillsthis long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The non-segmented negative-strand RNA viruses (order Mononegavirales)comprise several important human pathogens. The order of their genes,which is highly conserved, is the major determinant of the relativelevels of gene expression, since genes that are close to the singlepromoter site on the viral genome are transcribed at higher levels thanthose that occupy more distal positions. An infectious cDNA clone of theprototypic vesicular stomatitis virus (VSV) was manipulated to rearrangethe order of four of the five viral genes, while leaving all otheraspects of the viral nucleotide sequence unaltered. In one set of cDNAclones, the middle three genes (which encode the phosphoprotein P, thematrix protein M, and the glycoprotein G) were rearranged into all sixpossible orders. In another set, the gene for the nucleocapsid protein Nwas moved away from its wild-type promoter-proximal position and placedsecond, third or fourth. In a final rearrangement, the G protein gene,which encodes the major surface antigen and the target for neutralizingantibodies, was put next to the promoter, in the position for maximumexpression. Infectious viruses were recovered from each of theserearranged cDNAs and examined for their levels of gene expression andgrowth potential in cell culture, and their immunogenicity and virulencein mice. Rearrangement changed the expression levels of the encodedproteins and attenuated the viruses to different extents both incultured cells and in mice. Increasing the expression of the G proteinenhanced and accelerated the immune response in inoculated mice. Sincethe Mononegavirales do not undergo homologous recombination, generearrangement should be irreversible and thus provides a rational methodfor developing securely attenuated live vaccines against this type ofvirus.

In one embodiment of the present invention, there is provided a methodof increasing expression of a promoter distal gene in a virus of theorder Mononegavirales, comprising the step of rearranging gene order ofthe virus by moving the promoter distal gene toward a wild-type 3′promoter proximal position site.

In another embodiment of the present invention, there is provided arecombinant virus of the order Mononegavirales having a rearrangedgenome, wherein the genome is rearranged by moving a promoter distalgene of the virus toward a wild type 3′ promoter proximal position site.Such recombinant virus can be used for accelerating and enhancing aprotective immune response.

In still another embodiment of the present invention, there is provideda method of attenuating a virus of the order Mononegavirales byrearranging gene order of the virus by moving a gene away from its wildtype position, or by rearranging gene order of the virus by moving anessential limiting factor gene away from its wild type 3′ promoterproximal position site.

In yet another embodiment of the present invention, there is provided amethod of constructing an attenuated virus useful for a vaccine,comprising the steps of: rearranging the gene order of the virus bymoving a gene away from its wild-type 3′ promoter proximal positionsite, wherein the gene is an essential limiting factor for genomereplication; and placing a gene coding for an immune response inducingantigen in the position closest to the 3′ end of the gene order of thevirus.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the gene orders of the rearranged VSV genomes.

FIG. 2 shows the stepwise procedure for generation of rearranged VSVgenomic cDNAs.

FIG. 3A shows the cleavage specificity of restriction enzymes used togenerate cDNA modules for gene order rearrangement. Using PCR, either aBspMI or BsaI site is positioned at each end of the P, M and G genes ofVSV, and at the 3′ end of the N gene and the 5′ end of the L gene, suchthat the sticky ends correspond to 4 of the conserved nucleotides at theintercistronic junctions.

FIG. 3B shows fragments of VSV genome cloned for gene orderrearrangement.

FIG. 4 shows the strategy for construction of rearranged genomes N2, N3,N4 and G1N2.

FIG. 5A shows the schematic diagram of the VSV genome showing positionsof PCR primers that annealed to the N or L genes, respectively (shown bythe arrows) and restriction enzyme cleavage sites and predicted fragmentsizes.

FIG. 5B shows the products after digestion with indicated enzymes of thecDNAs of viral RNA from viruses GMP, MGP, PGM, PMG, GPM and MPG (lanes1-6, respectively). Fragments were analyzed by electrophoresis on a 1%agarose gel in the presence of ethidium bromide. Lane M=marker DNAfragments with sizes as indicated.

FIG. 6 shows viral RNAs synthesized in BHK-21 cells that were infectedwith the wild-type and variant viruses. Viral RNAs were labeled with[³H]uridine, resolved by electrophoresis on an agarose-urea gel, anddetected by fluorography. The infecting viruses are shown above thelanes, and the viral RNAs are identified on the left.

FIG. 7 shows viral proteins synthesized in BHK-21 cells that wereinfected with the wild-type and variant viruses. Viral proteins werelabeled with [³⁵S]methionine, resolved by electrophoresis on anSDS-polyacrylamide gel, and detected by autoradiography. The infectingviruses are shown above the lanes, and the viral proteins are identifiedon the left. Uninf, uninfected cells.

FIGS. 8A and 8B show the molar ratios of proteins synthesized in BHK-21cells that were infected with the wild type and variant viruses.Proteins were labeled, resolved on SDS-polyacrylamide gels as shown inFIG. 7, and quantitated by phosphorimaging. Molar ratios were calculatedafter normalizing for the methionine contents of the individualproteins: N-14, P-5, M-11, G-10 and L-60.

FIG. 9 shows the single step growth curves of wild type VSV and therearranged variants in BSC-1 cells. Viral titers were measured induplicate at each time point during three independent single-step growthexperiments at 37° C., and the results were averaged.

FIG. 10 shows pathogenesis of wild-type (wt) and variant virusesfollowing intranasal inoculation into mice. The time course of morbidity(gray bars) and mortality (black bars) in animals that receivedintranasal inoculation of 100 PFU of each of the variant viruses isshown. No further changes occurred after the time periods shown.

FIG. 11 shows the viral specific RNA synthesized in BHK-21 cellsinfected with rearranged viruses N1 (wt), N2, N3 and N4. Conditions ofinfection, labeling and analysis were as described in FIG. 6.

FIG. 12 shows the molar ratios of the VSV specific proteins synthesizedin BHK-21 cells following infection with rearranged viruses N1 (wt), N2,N3 and N4. Proteins were analyzed as described in FIG. 7 and molarratios calculated as described in FIG. 8.

FIG. 13 shows replication of viruses with N gene translocations bysingle step growth in BHK cells.

FIG. 14 shows relative lethality of viruses N1 (wt), N2, N3 and N4 formice.

FIG. 15 shows a comparison of antibody production and ability to protectagainst lethal challenge for viruses N1 (wt), N2, N3 and N4.

FIG. 16 shows the viral specific RNA synthesized in BHK-21 cellsinfected with viruses containing a foreign gene (I) inserted at each VSVintergenic junction. Conditions of infection, labeling and analysis areas described in FIG. 6 except the labeling time was from 2 to 4.5 hourspostinfection.

FIG. 17 shows the gene order of the variant viruses: N1G4 (wild type),G1N2, G3N4, and G1N4.

FIG. 18 shows synthesis of viral proteins in BHK-21 cells infected withthe variant viruses. In FIG. 18A, BHK-21 cells were infected at a MOI of50 and incubated at 37° C. for 5 hr in the presence of actinomycin D (5μg/ml) for the final 2 hr. Infected cells were then starved formethionine for 30 min and exposed to medium containing [³⁵S]methionine(30 μCi/ml) for 1 hr. Total infected cell proteins were analyzed bySDS-PAGE. In FIG. 18B, virions were isolated from supernatant fluids ofBHK-21 cells infected at a MOI of 5 and exposed to [³⁵S]methionine (50μCi/ml) from 2.5 to 12 hr post-infection. Virus particles were purifiedby centrifugation through 10% sucrose and their protein contentsdetermined by SDS-PAGE. Viral proteins shown in FIG. 18A and FIG. 18Bwere quantitated by phosphorimaging and expressed as molar percentagesof each viral protein in infected BHK-21 cells in FIG. 18C or molarpercentages of each protein in purified virions in FIG. 18D. Data shownaverage two independent experiments. Lanes: 1, N1G4 (wt); 2, G1N2; 3,G3N4; 4, G1N4; 5, uninfected cells.

FIG. 19 shows single-step growth analysis. Viruses were assayed fortheir ability to replicate by single-step growth in BHK-21 cells at 37°C. Cells were infected at a multiplicity of infection of 3 and samplesof the supernatant medium harvested at the indicated time points.Samples were titrated in duplicate by plaque assay on Vero-76 cells.Average virus yields per cell were determined at 24 hr post-infection(inset).

FIG. 20 shows pathogenesis in mice. The viruses shown were administeredintranasally to groups of 6 mice at a dose of 1,000 PFU per mouse, andthe animals were monitored daily for signs of morbidity and mortality.No further changes occurred after day 12.

FIG. 21 shows average weight of mice inoculated with the rearrangedviruses. Groups of 6 mice were inoculated intranasally with serial10-fold dilutions of N1G4 (wt), G1N2, G3N4, or G1N4 ranging from 10,000to 1 pfu/animal. Control mice received inoculation medium alone. Thevertical dotted line indicates day of challenge with 5.4×10⁶ pfu/mouseof wild-type virus. For each group, all living animals were weighedtogether and the average weight determined. , 10,000 pfu; {circle around(1)}, 1,000 pfu; , 100 pfu; , 10 pfu; ↑, 1 pfu; +, medium.

FIG. 22 shows kinetics of antibody production in response to inoculationwith the rearranged and wild-type viruses. Groups of 6 mice wereinoculated intranasally with serial 10-fold dilutions of N1G4 (wt),G1N2, G3N4, or G1N4 ranging from 10,000 to 1 pfu/animal. Control micereceived inoculation medium only. The vertical dotted line indicates theday of challenge with 5.4×10⁶ pfu/mouse of wild type virus. Serum wascollected by tail bleeds from 2-4 animals at weekly intervals, the serumpooled and the level of antibody raised against VSV determined bytitration on detergent-lysed VSV-infected cell antigen in an ELISA.Antibody levels are expressed as log₁₀ titers. , 10,000 pfu; {circlearound (1)}, 1,000 pfu; , 100 pfu; , 10 pfu; ↑, 1 pfu; +, medium.

FIG. 23 shows that groups of 6 mice were inoculated intranasally withserial 10-fold dilutions of N1G4 (wt), G1N2, G3N4, or G1N4 ranging from10,000 to 1 pfu/animal. Control mice received inoculation medium only.Mice were assessed for neutralizing antibody levels as measured in serumsamples on the day of challenge by plaque reduction assay (FIG. 23A).Neutralizing antibody levels are expressed as the reciprocal of thehighest dilution giving a 50% reduction in wild-type virus plaques onVero-76 cells. * sera from animals given 1 PFU or 10 PFU of N1G4 or G1N2virus had background levels of neutralizing antibody. Mice were alsoassayed for ability to survive intranasal challenge by 5.4×10⁶ PFU ofN1G4 virus (FIG. 23B). The dotted line shows the lethality of this dose(83%) in unvaccinated, age-matched, control animals 21 days afterchallenge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention illustrates that introduction of specific changesinto the genome of a negative strand RNA virus allowed translocation ofthe gene for the nucleocapsid (N) protein to successive positions on thegenome and demonstrated directly that the position of a gene relative tothe promoter determined the level of expression. Levels of N proteinsynthesis control the level of RNA replication. Consistent with this,the present invention demonstrates that as the level of N mRNA andprotein synthesis in cells infected with viruses N2, N3 and N4 wasreduced, the level of genomic RNA replication was also reduced.Correspondingly, the production of infectious virus in cell culture wasreduced in increments up to four orders of magnitude with virus N4.Finally, concomitant with reduced replication potential, the lethalityof viruses N2, N3, and N4 for mice following IN inoculation was reducedby approximately one, two or three orders of magnitude, respectively,compared to the wild-type virus.

These data demonstrate that translocating a single gene essential forreplication to successive positions down the viral genome lowered thegrowth potential in cell culture and the lethality of the viruses formice in a stepwise manner. However, the ability of the viruses to elicita protective immune response in mice was not altered in correspondencewith the reduction in virulence. Therefore, since the viruses allcontained the wild-type complement of genes and all were competent toreplicate, albeit at reduced levels, the level of replication wassufficient to induce a protective host response. Thus, for somerearranged viruses, the protective dose and the lethal dose were 1,000fold different, in contrast to the situation with wild-type virus wherethe lethal dose and protective dose overlap. Taken together, these datasuggest a means of attenuating non-segmented negative strand RNA virusesin a predictable, incremental manner that would allow one to determinean optimal level of attenuation to avoid disease production without lossof replication potential to induce a sufficient immune response.

Since the Mononegavirales have not been observed to undergo homologousrecombination, gene rearrangement is predicted to be irreversible, andtherefore, the present invention provides a rational, alternative methodfor developing stably attenuated live vaccines against the non-segmentednegative strand RNA viruses. Furthermore, based on the close similarityof genome organization and control of gene expression, this approach togenerating attenuated viruses should be applicable to the entire familyof Mononegavirales, which includes the Rhabdoviridae, such as rabies,the Paramyxoviridae, such as measles, mumps, respiratory syncytialvirus, and parainfluenza viruses I-IV, and the Filoviridae such as Ebolaand Marburg viruses. These represent some of the most problematic viralpathogens extent.

In one embodiment of the present invention, there is provided a methodof increasing expression of a promoter distal gene in a virus of theorder Mononegavirales, comprising the step of rearranging gene order ofthe virus by moving the promoter distal gene toward a wild-type 3′promoter proximal position site. Preferably, the distal gene encodes asurface glycoprotein. For vesicular stomatitis virus, one distal genethat encodes a surface glycoprotein is the gene for the attachmentglycoprotein G. For respiratory syncytial virus, one distal gene thatencodes a surface glycoprotein is referred to as the attachmentglycoprotein (G) gene; another distal gene that encodes a surfaceglycoprotein is the respiratory syncytial virus fusion (F) protein gene.For the measles virus, the distal gene that encodes a surfaceglycoprotein is referred to as the H (hemagglutinin) gene. For the mumpsand parainfluenza viruses, the distal gene that encodes a surfaceglycoprotein is referred to as the HN (hemagglutinin/neuraminidase)gene. A person having ordinary skill in this art would readilyrecognize, for each specific virus of the order Mononegavirales, whichdistal gene that encodes a surface glycoprotein would be manipulated inorder to perform the methods of the present invention.

In another embodiment of the present invention, there is provided arecombinant virus of the order Mononegavirales having a rearrangedgenome, wherein the genome is rearranged by moving a promoter distalgene of the virus toward a wild type 3′ promoter proximal position site.Such recombinant virus can be used for accelerating and enhancing aprotective immune response.

In still another embodiment of the present invention, there is provideda method of attenuating a virus of the order Mononegavirales byrearranging gene order of the virus by moving a gene away from its wildtype position, or by rearranging gene order of the virus by moving anessential limiting factor gene away from its wild type 3′ promoterproximal position site. Preferably, the gene is placed in the next tolast position in the gene order of the virus. Furthermore, it ispreferable that the gene which is an essential limiting factor forgenome replication is the nucleocapsid (N) gene. Representative examplesof viruses of the order Mononegavirales are a Rhabdovirus, such asrabies virus or vesicular stomatitis virus, a Paramyxovirus, such asmeasles, mumps, parainfluenza virus or respiratory syncytial virus(human and bovine), or a Filovirus, such as Ebola virus or Marburgvirus. The present invention also includes a virus attenuated accordingto this method.

In yet another embodiment of the present invention, there is provided amethod of constructing an attenuated virus useful for a vaccine,comprising the steps of rearranging gene order of the virus by moving agene away from its wild-type 3′ promoter proximal position site, whereinthe gene is an essential limiting factor for genome replication; andplacing a gene coding for an immune response inducing antigen in theposition closest to the 3′ end of the gene order of the virus.Preferably, the essential limiting factor gene is the nucleocapsid (N)gene and the gene is placed in the next to last position in the geneorder of the virus. Still preferably, the gene coding for an immuneresponse inducing antigen may be the attachment glycoprotein (G) gene, afusion gene or the hemagglutinin/neuraminidase gene. A person havingordinary skill in this art would be able to readily substitute suitableimmune response-inducing antigens. The present invention also includes avirus attenuated according to this method.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Therefore, if appearing herein, the following terms shall havethe definitions set out below.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the non-transcribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo; i.e., capable of replication under its own control.An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis. An “expression control sequence” is a DNAsequence that controls and regulates the transcription and translationof another DNA sequence. A coding sequence is “operably linked” and“under the control” of transcriptional and translational controlsequences in a cell when RNA polymerase transcribes the coding sequenceinto mRNA, which is then translated into the protein encoded by thecoding sequence.

In general, expression vectors containing promoter sequences whichfacilitate the efficient transcription and translation of the insertedDNA fragment are used in connection with the host. The expression vectortypically contains an origin of replication, promoter(s), terminator(s),as well as specific genes which are capable of providing phenotypicselection in transformed cells. The transformed hosts can be fermentedand cultured according to means known in the art to achieve optimal cellgrowth.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. A “cDNA” is defined ascopy-DNA or complementary-DNA, and is a product of a reversetranscription reaction from an mRNA molecule. An “exon” is an expressedsequence transcribed from the gene locus, whereas an “intron” is anon-expressed sequence that is from the gene locus.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell. A “cis-element” is a nucleotide sequence, alsotermed a “consensus sequence” or “motif”, that interacts with otherproteins which can upregulate or downregulate expression of a specificgene locus. A “signal sequence” can also be included with the codingsequence. This sequence encodes a signal peptide, N-terminal to thepolypeptide, that communicates to the host cell and directs thepolypeptide to the appropriate cellular location. Signal sequences canbe found associated with a variety of proteins native to prokaryotes andeukaryotes.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters containShine-Delgarno sequences in addition to the −10 and −35 consensussequences.

The term “oligonucleotide” is defined as a molecule comprised of two ormore deoxyribonucleotides, preferably more than three. Its exact sizewill depend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide. The term “primer” as usedherein refers to an oligonucleotide, whether occurring naturally as in apurified restriction digest or produced synthetically, which is capableof acting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension product, which iscomplementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be eithersingle-stranded or double-stranded and must be sufficiently long toprime the synthesis of the desired extension product in the presence ofthe inducing agent. The exact length of the primer will depend upon manyfactors, including temperature, source of primer and use the method. Forexample, for diagnostic applications, depending on the complexity of thetarget sequence, the oligonucleotide primer typically contains 15-25 ormore nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence or hybridize therewith andthereby form the template for the synthesis of the extension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to enzymes which cut double-stranded DNA at or near aspecific nucleotide sequence. “Recombinant DNA technology” refers totechniques for uniting two heterologous DNA molecules, usually as aresult of in vitro ligation of DNAs from different organisms.Recombinant DNA molecules are commonly produced by experiments ingenetic engineering. Synonymous terms include “gene splicing”,“molecular cloning” and “genetic engineering”. The product of thesemanipulations results in a “recombinant” or “recombinant molecule”.

A cell has been “transformed” or “transfected” with exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a vector or plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations. An organism,such as a plant or animal, that has been transformed with exogenous DNAis termed “transgenic”.

As used herein, the term “host” is meant to include not only prokaryotesbut also eukaryotes such as yeast, plant and animal cells. A recombinantDNA or RNA molecule or gene of the present invention can be used totransform a host using any of the techniques commonly known to those ofordinary skill in the art. One preferred embodiment is the use of avectors containing coding sequences for the RNA molecules or cDNAmolecules of the present invention for purposes of transformation.Prokaryotic hosts may include E. coli, S. typhimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts suchas Pichia pastoris, mammalian cells and insect cells, and morepreferentially, plant cells, such as Arabidopsis thaliana and Tobaccumnicotiana.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90%or 95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. In another example, the coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., a cDNA wherethe genomic coding sequence contains introns, or synthetic sequenceshaving codons different than the native gene). Allelic variations ornaturally-occurring mutational events do not give rise to a heterologousregion of DNA as defined herein.

A standard Northern blot assay can be used to ascertain the relativeamounts of mRNA in a cell or tissue obtained from plant or othertransgenic tissue, in accordance with conventional Northernhybridization techniques known to those persons of ordinary skill in theart. Alternatively, a standard Southern blot assay may be used toconfirm the presence and the copy number of the gene in transgenicsystems, in accordance with conventional Southern hybridizationtechniques known to those of ordinary skill in the art. Both theNorthern blot and Southern blot use a hybridization probe, e.g.radiolabeled cDNA, either containing the full-length, single strandedDNA or a fragment of that DNA sequence at least 20 (preferably at least30, more preferably at least 50, and most preferably at least 100consecutive nucleotides in length). The DNA hybridization probe can belabeled by any of the many different methods known to those skilled inthis art. Alternatively, the label may be incorporated directly into theRNA or protein molecule by many different methods known to those ofskill in this art.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Proteins canalso be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. The preferred isotope may be selected from ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090,3,850,752, and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

As used herein, the term “attenuation” is defined as either a geneticmechanism involving premature termination of transcription used toregulate expression of a gene, or immunologically, the process whereby apathogenic microorganism loses its virulence.

As used herein, the term “lethal dose” is defined as the amount of virusinoculum required to confer lethality on the host.

As used herein, the term “protective dose” is defined as the amount ofvirus inoculum that produces a sufficient immune response towards thevirus without resulting in lethality.

As used herein, the term “rearrangement” is defined as the reordering ofthe genes within the viral genome, such that the gene and the intergenicregions remain wild-type and only the order with respect to the 3′terminus is altered.

As used herein, the term “negative strand RNA virus” is defined as aclassification of RNA viruses in which the genome comprises the negativestrand of an RNA molecule.

The present invention also demonstrates that it is possible to increasethe expression of a promoter distal gene, e.g., the G gene, whichencodes the attachment glycoprotein, by moving it to a promoter proximalsite. To show that an increase in the production of the G protein duringinfection could elicit a greater protective immune response, changeswere engineered into an infectious cDNA clone of the VSV genome and twonovel viruses were recovered in which the glycoprotein gene was movedfrom its normal fourth position to the first position in the gene order.One virus had the gene order 3′-G-N-P-M-L-5′ (G1N2) and the second3′-G-P-M-N-L-5′ (G1N4). The in vitro and in vivo characteristics ofthese viruses were assessed and compared to those of viruses having thegene orders 3′-P-M-G-N-L-5′ (G3N4) and 3′-N-P-M-G-L-5′ (N1G4), thelatter being the wild-type gene order. Differences were observed in thereplication of these viruses in cell culture, lethality in mice,kinetics and levels of antibody production, and their ability to protectagainst challenge with a lethal dose of VSV.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

Example 1 Viruses and Cells

The San Juan isolate of the Indiana serotype of VSV provided theoriginal template for most of the cDNA clones used herein. However, thegene encoding the G protein was originally derived from the Orsayisolate of VSV Indiana (Whelan et al., 1995). Baby hamster kidney(BHK-21) cells were used to recover viruses from cDNAs and for singlestep growth experiments and radioisotopic labeling of RNAs and proteins.African green monkey kidney (BSC-1 and BSC-40) cells were used forplaque assays.

Example 2 Plasmid Construction and Recovery of Infectious Viruses

Each of the five genes of VSV is flanked by a common sequence ofeighteen nucleotides. Thus, it was possible to construct individualmolecular clones from which DNA fragments precisely encompassing eachgene could be released by digestion with an appropriate restrictionendonuclease. Restriction endonucleases that cut at site remote fromtheir recognition sequences were used to create gene segments havingcohesive ends that corresponded to the same four nucleotides (ACAG) ofthe conserved intercistronic regions. In this way, the DNA segments thatencompassed each of the five genes could be reassembled in any desiredorder to create a family of DNA plasmids whose nucleotide sequencescorresponded precisely to that of wild-type VSV, except for the factthat their genes were rearranged. A diagram of the steps involved in theconstruction of the rearranged virus genomes N1 (wt), GMP, MGP, PGM,GPM, MPG, N2, N3, N4, G1N2 and G1N4 is shown in FIGS. 2, 3A, 3B, and 4.

Infectious viruses were recovered from these DNA plasmids by methodsdescribed (Whelan et al., 1995). Briefly, BHK cells were infected withthe vaccinia virus recombinant that expresses T7 RNA polymerase, VTF7-3,(Fuerst et al., 1986) and cotransfected with one of the rearranged cDNAplasmids and the three support plasmids that express the N, P and Lproteins required for RNA encapsidation and replication. Infectiousviruses were recovered from the supernatant of transfected cells,amplified by low-multiplicity passage on BHK-21 cells, and filteredthrough 0.2 mm filters to remove contaminating VTF7-3. The gene ordersof the recovered viruses were verified by amplifying the rearrangedportions of the viral genomes using reverse transcription and polymerasechain reaction (PCR) followed by restriction enzyme analysis with a setof enzymes which distinguished the rearranged gene orders (FIGS. 5A and5B).

Example 3 Single-cycle Virus Replication

Monolayer cultures of 10⁶ BHK-21, BSC-40 or BSC-1 cells were infectedwith individual viruses at an input multiplicity of 3. Following a onehour adsorption period, the inoculum was removed, cultures were washedtwice, fresh media was added and cultures were incubated at 31° C. or37° C. Samples were harvested at the indicated intervals over a 36 hourperiod and viral replication quantitated by plaque assay on confluentmonolayers of BSC-40 cells.

Example 4 Analysis of Viral RNA and Protein Synthesis

Confluent monolayer cultures of BHK-21 cells were infected withindividual viruses at an input multiplicity of 5 PFU per cell and givena one hour adsorption period. For analysis of viral RNA synthesis,cultures were treated with actinomycin D (5 μg/ml) at 1.5 hourspost-infection for 30 minutes prior to addition of [³H]-uridine (30μCi/ml) for a 2 or 4 hour labeling period. Cells were harvested,cytoplasmic extracts prepared and RNA analyzed on 1.75% agarose-ureagels as described (Pattnaik and Wertz, 1990). Protein synthesis wasanalyzed at four hours post-infection by addition of [³⁵S]-methionine(40 μCi/ml) for a 30 minute labeling period following a 30 minuteincubation in methionine free media. Cytoplasmic extracts were preparedand proteins analyzed on 10% polyacrylamide gels as described previously(Pattnaik and Wertz, 1990). Individual RNAs or proteins were quantitatedby densitometric analysis of autoradiographs using a Howteck Scanmaster3 with Pdi Quantity One software and molar ratios were subsequentlycalculated.

Example 5 Virulence in Mice

The lethality of individual viruses was measured in male Swiss-Webstermice, 3-4 weeks old, obtained from Taconic Farms. Groups of 5-6 lightlyanesthetized (Ketamine/Xylazine) animals were inoculated with diluent(PBS) or with serial ten-fold dilutions of individual viruses by eitherthe intracranial route in a volume of 30 μl or by the intranasal routein a volume of 15 μl. Animals were observed daily and the 50% lethaldose (LD₅₀) for each virus was calculated by the method of Reed andMuench (1938).

Example 6 Protection of Mice

Groups of control mice inoculated with diluent or inoculatedintranasally with non-lethal doses of individual viruses were monitoredby tail bleeds for neutralizing serum antibody production. On day 14post-inoculation, mice were challenged with 1.3×10⁶ PFU of wild-typevirus (designated N1) administered intranasally in 15 μl while underlight anesthesia as above. Challenged animals were observed for 21 days.

Example 7 A General Approach to Rearranging the Genes of theMononegavirales

To rearrange the genes of VSV without introducing any other changes intothe viral genome, the polymerase chain reaction (PCR) was used toconstruct individual cDNA clones of the N, P, M, and G genes flanked bysites for restriction enzymes that cut outside their recognitionsequences. To flank the P, M, and G genes, BspM1 sites were used,whereas to flank the N gene, Bsa1 sites were used (N contains aninternal BspM1 site). PCR primers were designed to position theserestriction sites so that the four-base cohesive ends left afterendonuclease digestion corresponded to the ACAG sequence of theconserved 5′ AACAG . . . 3′ that occurs at the start of each VSV mRNA(see also FIG. 3A). For example: 5′ . . . ACCTGCACT AACAG . . .AAAAAAACTAACAGAGATGCAGGT . . . 3′ (SEQ ID No. 1), where the VSVsequence, written in the positive sense, is in italics, the BspM1recognition sites are in bold letters, and the four-base cohesive endsleft by BspM1 digestion are underlined. In this way, the four genes,together with their respective intergenic junctions, were recovered onindividual DNA fragments that had compatible cohesive termini (FIGS. 3Aand 3B). The only deliberate departure from the wild-type sequence wasthat the untranscribed intergenic dinucleotide was made 5′-CT-3′ at alljunctions, including that following the P gene where the wild-typesequence is 5′-GT-3′. This mutation is apparently silent (Barr et al.,1997). To circumvent the effect of spurious mutations arising duringPCR, the termini of the cloned genes were sequenced and their interiorswere replaced with corresponding DNA fragments from the infectiousclone.

Two other starting plasmids were required to reconstruct the rearrangedfull-length clones: one contained a bacteriophage T7 promoter followedby the VSV leader sequence, with a unique BspM1 site positioned to cutwithin the 5′ (A)ACAG at the start of the N gene: 5′ . . .GAAACTTTAACAGTAATGCAGGT . . . 3′ (SEQ ID No. 2). The other plasmidcontained the first 420 nucleotides of the L gene and had a unique BspM1site positioned to cut within the same sequence at the start of L: 5′ .. . ACCTGCACTAAC AGCAATCATG . . . 3′ (SEQ ID No. 3). The N, P, M and Ggene fragments were ligated unidirectionally into the unique BspM1 sitesof these plasmids to rebuild the viral genome in a stepwise manner fromeither the 3′ or the 5′ end. Insertion of each gene recreated awild-type intergenic junction and left a unique BspM1 site to receivethe next gene.

The final step of plasmid construction was to add a DNA fragment fromthe infectious clone that encompassed the remaining 6 kb of the L gene,the 5′ end of the viral genome, and the ribozyme and T7 terminator thatare needed for the intracellular synthesis of replication-competenttranscripts (Pattnaik et al., 1992). This approach can be applied to anyof the Mononegavirales which have conserved sequences at theirintergenic junctions. The rearranged gene orders that were created inthis manner are shown in FIG. 1. To validate this cloning strategy andto verify that the individual genes encoded functional proteins, aplasmid that contained the wild-type genome was created in parallel withthe rearranged cDNA clones. Virus recovered from this plasmid was usedas the wild-type (N1, see FIG. 1). In all cases, the conserved 23nucleotide intergenic region was maintained between genes.

Example 8 Generation of Viruses with Rearranged Genomes

Initial rearrangements of the cDNA of the genome of VSV wereconservative, in light of the highly conserved nature of the genomes ofall viruses in the family Mononegavirales, and the knowledge thatprecise molar ratios of the VSV nucleocapsid (N) protein, phosphoprotein(P) and RNA polymerase (L) protein are required for replication. The 3′most gene, N, and the 5′ most gene, L, were originally maintained intheir natural positions and the three central genes of VSV, the P, M andG genes, were rearranged in all possible combinations to generate the 6genome orders (N1 (wt), GMP, MGP, PGM, GPM and MPG) as shown in FIG. 1.The wild-type gene order, N1, was generated as described above to serveas a test that all of the cDNA elements were functional. Each of thecDNAs was constructed in a specialized T7 expression plasmid designed togenerate RNAs having precise 5′ and 3′ termini (Pattnaik et al., 1992).

The ability of the rearranged cDNAs to generate a functional RNA genomewas demonstrated by transfecting each of the six rearranged cDNAs intoBHK cells infected with vaccinia virus expressing the T7 polymerase(Fuerst et al., 1986) concomitantly with cDNA clones encoding the VSV N,P, and L proteins to encapsidate the RNA transcribed from the cDNAclones and to form functional ribonucleocapsids as described (Whelan etal., 1995). Virus was recovered with varying efficiency from all six ofthe cDNA constructs and amplified in the presence of cytosinearabinoside (25 μg/ml) following filtration through 0.2 μm filters toremove the recombinant vaccinia virus used to express the T7 polyrneraserequired for transcription of the cDNAs to yield RNA virus.

Example 9 The Gene Order of the Recovered RNA Viruses Reflects That ofthe cDNA From Which They Were Generated

The gene orders of the recovered viruses were determined after threepassages in cell culture by amplifying a 4.1-kb fragment encompassingthe rearranged portions of the viral genomes by reverse transcriptionand PCR, followed by restriction enzyme analysis of the PCR products.PCR was carried out with primers located in the N and L genes. Aftercleavage with restriction endonuclease AccI, BglI, or PstI, which cleaveuniquely in the P, M, or G gene, respectively, the observed sizes of thedigestion products were found to be exactly as predicted (FIGS. 5A and5B). The data showed that the gene orders of the recovered virusescorresponded to the cDNA clones from which there were recovered. Therewas no evidence for the reappearance of the wild-type gene order amongthe variants.

Example 10 Synthesis of Viral RNAs and Proteins

The recovered viruses were next examined for their levels of geneexpression. Synthesis of viral RNAs and proteins by the variant viruseswas examined by metabolic incorporation of [³H]uridine or[³⁵S]methionine into infected cells, and analysis of the radiolabeledproducts by gel electrophoresis. The same species of viral RNAs weremade in cells infected with the wild-type virus and with each of thevariants: the 11.16-kb genomic RNA and mRNAs representing the L, G, N,P, and M genes (FIG. 6). The latter two mRNAs are similar in size andcomigrated during electrophoresis. No novel or aberrant RNA species werefound in cells infected with the variant viruses, showing that the viruspreparations were free of DI particles (FIG. 6). Moreover, thesimilarities among the RNA patterns reinforced the idea that thebehavior of the viral polymerase during transcription across theintergenic junctions was determined exclusively by local sequenceelements at these positions, with no detectable long-range influences.In accordance with the RNA patterns, the viral proteins made by thevariant viruses also resembled qualitatively those made during wild-typeinfection (FIG. 7).

Although the RNA and protein profiles of cells infected with thewild-type and variant viruses were qualitatively similar, measurement ofthe relative levels of the different RNAs and proteins showed that thevariant viruses expressed their genes in molar ratios that differed fromboth the wild type and one another (FIGS. 8A and 8B). Normalizing theexpression of each protein to that of the promoter-proximal N gene foreach variant virus showed that the relative expression level of a genedepended primarily on its location in the genome and thus on itsdistance from the promoter, just as predicted by the model ofprogressive transcriptional attenuation. This is clearly exemplified bycomparison of the molar rations of proteins expressed by the wild-type(PMG) with variant GMP in which the order of the internal genes isreversed (FIGS. 8A and 8B). A similar quantitative analysis of the mRNAprofiles was complicated by the lack of resolution of the M and P mRNAs(FIG. 6), but measurement of the L, G, and N mRNA levels reinforced theconclusion that the proximity of a gene to 3′ end of the viral genomewas the major determinant of its level of transcription. For example,the differences in the relative abundance of G mRNA reflected theposition of the G gene (FIG. 6). The RNA profiles also showed that thelevel of RNA replication, as measured by the abundance of the 11.1-kbgenomic and antigenomic RNAs, differed substantially among the variants(FIG. 6).

Example 11 Replication of Viruses with Rearranged Genomes

The variant viruses were compared for their ability to replicate underthe conditions of plaque formation and single-cycle growth. Althoughsome of the viruses such as MGP and MPG were indistinguishable from theN1 wild type virus (PMG) in these assays, others such as GMP, GPM, andPGM formed significantly smaller plaques than the wild type onmonolayers of BSC-1 cells (Table 1). Moreover, GMP plaques ceased togrow after 24 h when those of the wild type virus and the other variantswere still increasing in size (Table 1). The impaired replication ofGMP, GPM, and PGM was also demonstrated during single-cycle growth onBSC-1 cells (FIG. 9). At 17 h post-infection, the incremental yields ofthe variants averaged over three independent growths and expressed aspercentages of the wild type were as follows: for MGP, 107%; for MPG,51%; for GMP, 23%; for PGM, 21%; and for GPM, 1.6% (Table 2).

TABLE 1 Plaque diameter (mean ± standard error)^(a) Virus 24 h 30 h PMG(wild type) 4.02 ± 0.12 4.81 ± 0.19 GMP 3.08 ± 0.17 3.10 ± 0.18 MGP 3.96± 0.19 4.97 ± 0.18 PGM 3.36 ± 0.12 3.86 ± 0.14 GPM 2.26 ± 0.09 3.16 ±0.13 MPG 3.85 ± 0.18 5.43 ± 0.17 ^(a)Plaque diameters were measured fromphotographs taken at approximately two-fold magnification of groups of50 (24 h) or 70 (30 h) viral plaques formed at 37° C. on monolayers ofBSC-1 cells.

Example 12 Virulence in Mice

Intracerebral or intranasal inoculation of wild-type VSV into micecauses fatal encephalitis. Since 1938, when Sabin and Olitsky firstdescribed the neuropathology and comparative susceptibility of mice toVSV encephalitis as a function of age and route of inoculation, youngmice have served as a convenient and sensitive small animal model forcomparing the lethality of VSV and its mutants (Sabin and Olitsky. 1938;Wagner, 1974). The pathogenesis of the variant viruses in mice wastherefore examined.

Intranasal inoculation of wild-type VSV into 3-4 week old mice causesencephalitis, paralysis and death after 7-11 days (Sabin and Olitsky,1938), with the LD₅₀ dose being about 10 PFU. The virulence of thevariant viruses was compared by inoculating groups of mice intranasallywith serial 10-fold dilutions ranging from 0.1 to 1,000 PFU per dose andobserving them twice daily. Viral gene orders were verified on virusesrecovered shortly after death from the brains of inoculated mice byusing the methodology shown in FIGS. 5A and 5B. In each case, the geneorder of the recovered virus corresponded to that of the inoculum (datanot shown).

The LD₅₀ doses for the variant viruses were similar to that of the wildtype, with viruses GPM, GMP, and MGP requiring slightly higher (1.5- to2-fold) dose (Table 2). These experiments were repeated three times, andthe results of a representative experiment show the time of appearanceof illness and death at a dose of 100 PFU per mouse (FIG. 10). Thewild-type infected animals first appeared sick at 6 dayspost-inoculation, rapidly became paralyzed, and died within two weeks.Recombinants GMP and MGP elicited reproducibly faster pathogenesis, withsymptoms developing 24-36 h earlier than in wild-type infected animals,whereas the onset of death from infection with MPG and GPM occurred 24to 36 h later (FIG. 10). In general, the paralysis that is typical ofinfection with wild-type VSV was less apparent with the variant viruses,but there was no evidence of persistent nervous system disease such asthat produced by some M protein mutants (Barr et al., 1997).

Virulence in mice could not be predicted from the cell culturephenotypes of the variant viruses (Table 2). Of the three recombinantswhose replication in cell culture was most compromised (GMP, PGM, andGPM), one (GPM) required 2 fold more virus for an LD₅₀ than thewild-type and showed slightly delayed killing in mice, whereas GMPinduced faster onset of symptoms and death, and PGM wasindistinguishable from wild-type. This lack of correlation between thebehavior of viruses in cell culture and their properties in animals is afamiliar observation among different animal viruses, but is interestingin this context where the only differences between the viruses were therelative levels of wild-type proteins that they expressed.

TABLE 2 Summary of properties of variant viruses Gene Relative RelativeLD₅₀ Onset of order plaque size^(a) burst size^(b) value^(c)symptoms^(d) PMG (wt) 1.00 1.00 14 6.0 GMP 0.64 0.23 21 4.5 MGP 1.031.07 21 5.5 PGM 0.80 0.21 12 5.5 GPM 0.66 0.016 30 5.5 MPG 1.13 0.51 115.5 ^(a)Measured at 30 h post-infection (see Table 1). ^(b)Measured at17 h post-infection (see FIG. 9). ^(c)PFU per mouse inoculatedintranasally. ^(d)Days after intranasal inoculation of 100 PFU per mouse(see FIG. 10).

Example 13 Effect of Severe Rearrangements on Recovery of Viable Virus

Encouraged by the relative tolerance that VSV exhibited forrearrangement of the three internal genes based on recovery ofinfectious virus, further rearrangements were made that altered theposition of the gene for the nucleocapsid protein, N. The N protein isrequired in stoichiometric quantities to support encapsidation ofnascent genomic RNA during RNA replication (Patton et al., 1984). RNAreplication is dependent on constant synthesis of the N protein, andinhibition of N protein synthesis results in cessation of replication.If the level of N protein synthesis were lowered by moving the N geneprogressively away from its promoter proximal site (and thus loweringthe level of N gene expression), it would therefore result in loweredlevels of genomic replication. As such, the genome of VSV was altered atthe cDNA level by moving the N gene from the 3′ most position, whichresults in synthesis of the largest amount of N mRNA, to each sequentialinternal position as shown in FIG. 1 to create N2 (PNMGL), N3 (PMNGL),and N4 (PMGNL). N1 corresponds to the wild-type arrangement. A fourthand fifth variation, in which the G gene was moved from next to last inthe order and placed in front of the N gene, were also generated (FIG.1). This results in G1N2 (GNPML), as well as G1N4 (GPMNL), where theposition of the G and N genes were exchanged.

The cDNAs for N1-N4 and G1N2 and G1N4 were transfected into cells asdescribed above and analyzed for the ability to generate viable virus.Virus was recovered with comparative ease from N2, N3 and G1N2. Viruswas not recovered from N4 and G1N4, even with repeated trials usingstandard transfection conditions at 37° C. Virus corresponding to N4 andG1N4 was recovered by lowering the temperature of the transfections andsubsequent passages to 31° C.

Example 14 RNA Synthesis by Viruses with N Gene Rearrangements

Moving the N gene sequentially down the genome had a marked effect onthe level of replication and N mRNA synthesis (FIG. 11). The level of NmRNA synthesis decreased substantially from wild-type levels as the Ngene was moved successively away from the promoter in viruses N2, N3 andN4 (36%, 6% and 3% of wild-type, respectively; FIG. 11). Consistent withthis, an increase in the amount of G mRNA was observed with virus N4, inwhich the G gene was moved one position closer to the promoter as the Ngene replaced it as next to last in the gene order (FIG. 11). The amountof genomic RNA replication of N2, N3 and N4 declined relative towild-type (50%, 28% and 4%, respectively; FIG. 11), concomitant with thelowered expression of the N gene, as predicted if N protein synthesiswas limiting for replication. The overall level of transcription wasreduced also as the N gene was moved progressively promoter distal,presumably as a secondary effect due to the lowered number of genomictemplates.

Example 15 Protein Synthesis of Viruses with the N Gene Rearranged

All five of the VSV proteins were expressed in cells infected with therearranged viruses and they all co-migrated with those of the wild-typevirus. However, N protein synthesis declined as its gene was moved awayfrom the 3′ position. The data presented in FIG. 12 show how the molaramounts of the proteins decrease as a function of their distance fromthe 3′ terminus in the wild-type virus N1. When the N gene wastranslocated, the data in FIG. 12 show that the molar ratios of the Nprotein relative to the phosphoprotein P decreased progressively as theN gene was moved from first to second, third, or fourth in the geneorder. These results confirm the predictions from previous analysis ofgene expression in VSV and the sequential nature of transcription.Moreover, these data demonstrate directly that the position of a genedetermines its level of expression. Examination of the levels ofproteins in isolated, mature N1-N4 virions showed that the relativemolar ratios of the proteins in mature virus particles remainedessentially the same as that of the wild-type virus. However, lessoverall virus was produced from infections of N2-4, correlating with thelowered level of genomic RNA replication.

Example 16 Replication Ability in Cell Culture

Viruses with the N gene rearrangements replicated progressively lesswell as the N gene was moved downstream of its normal promoter proximalposition. Growth potential was analyzed by single step growth curves. N2and G1N2 were reduced in viral yields by approximately 15-fold at 37° C;N3 was reduced by 50 fold and N4 was reduced by 20,000 fold inreplication ability as compared to the wild-type virus (FIG. 13).Comparison of virus growth at 31° C. showed a similar progressivedecline, however, the effect was less pronounced than at 37° C., andoverall, this temperature was more permissive for growth (FIG. 13,inset). At 31° C., N4 replication was reduced approximately 100 foldcompared to wild-type. The burst size in PFU per cell for each of theviruses at 31° C. and 37° C., shows that the yield per cell declined inastepwise manner as the N gene was moved to each successive positiondown the genome (FIG. 13). The relative plaque sizes of the viruses alsovaried; plaques of N4 are compared to that of wild-type (<0.5 mmcompared to 3 mm in diameter at 42 hours post infection). These dataindicate that although the genes of N2, N3 and N4 were wild type,rearrangement of the genes and the subsequent alterations of the proteinmolar ratios rendered some step of the viral replication processpartially temperature sensitive.

Example 17 Lethality in Mice

Growth of VSV in mice, neuropathology and susceptibility to encephalitisby intracerebral or intranasal inoculation of wild-type, temperaturesensitive or plaque size variant viruses has been described in detail(Sabin and Olitsky, 1937; Shechmeister et al., 1967; Wagner, 1974;Youngner and Wertz, 1968). The lethality of viruses N2, N3 and N4 formice was examined in comparison with the wild-type virus N1 for both theintracerebral and intranasal routes of inoculation. The amounts of virusrequired for a lethal dose (LD₅₀) by each route is shown in Table 3. Byintercerebral inoculation, the LD₅₀ dose for each of the viruses was 1to 5 pfu, although the average time to death was about twice as longwith the N4 virus. These data show that when injected directly into thebrain, thereby circumventing the majority of host defenses, therearranged viruses eventually could cause fatal encephalitis.

Intranasal inoculation, by contrast, showed striking differences in theamount of virus required for a lethal dose (Table 3). Whereas the LD₅₀dose for the wild-type virus by IN administration was approximately 10pfu, the values for N2, N3 and N4 viruses were progressively greater. N2required 20 fold more virus, N3, 500 fold more virus, and N4 required3000 fold more virus than wild-type, i.e. 30,000 PFU for the LD₅₀. Thetime to onset of sickness (ruffled fur, lethargy, hind limb paralysis)and extent of death increased progressively compared to wild-typefollowing infection with viruses N2, N3 and N4 (FIG. 14) and the extentof mortality was a function of dose (Table 3). These data show that whenadministered by a peripheral route, the progressive reduction in virusreplication observed in cell culture correlated with a reduced lethalityin mice.

TABLE 3 Lethality of wild-type or Rearranged VSV Viruses for Mice LD₅₀Data* pfu/mouse (Average days to death) Intracranial Intranasal N1 NPMGL(WT) 1 (3-6) 11 (5-10) N2 PNMGL 5 (3-7) 250# (9-12) N3 PMNGL 5 (3-8)5,400# (7-9) N4 PMGNL 1 (4-11) 30,000 (10-12) *The LD₅₀ for each routeof inoculation was calculated from mortality among groups of 5 to 7 miceinoculated either IC or IN with five serial10-fold dilutions of virus.Data from a single internally controlled experiment are shown; theduplicate experiments carried out for each route of administration weresimilar. #Mortality data for this virus yielded a bell shaped deathcurve; the LD₅₀ dose was calculated from the lower part of the curve.Days to death are shown in parentheses.

Example 18 Ability of Rearranged Viruses to Protect Against Wild-typeChallenge

The observation that all of the viruses were lethal when inoculated ICindicated that even the most attenuated viruses were able to replicatein mice. This, coupled with the attenuation observed followingintranasal administration, raised the possibility that the attenuatedviruses might nevertheless be able to elicit a protective immuneresponse. To test this possibility, mice were immunized by INinoculation with serial ten-fold dilutions of the wild-type N1 or withvariant viruses N2, N3 or N4. The surviving animals were challenged 14days later by IN inoculation with 1.3×10⁶ PFU of wild-type virus. Thepercentage of animals surviving the challenge was a function of theimmunizing dose in agreement with previous studies (Wagner, 1974). Forviruses N2, N3 and N4, 300 PFU per mouse was the lowest dose giving 100%survival; 30 PFU yielded 80-90% survival; 3-6 PFU gave 45-85% survival;and doses below 3-6 PFU per mouse gave results that were notsignificantly different from those of age matched unimmunized controls(FIG. 15, dotted line in panel A). With the wild-type virus, the lethaldose and the protective dose were close, but in general, 80-85% ofanimals that survived administration of 3-6 PFU of virus were protected.

Measurement of serum antibody prior to challenge on day 14 showed thatdespite attenuation for virulence in mice, the level of neutralizingantibody present in the serum of animals immunized with viruses N2, N3and N4 was higher than that observed in the animals survivinginoculation of 3-6 PFU of wild-type virus and generally increased in adose dependent manner (FIG. 15B). The lethality of the wild-type virusprevented direct comparison of antibody titers at higher doses, however,the neutralizing antibody titers in animals both vaccinated with virusesN1-N4 and then challenged with 1×10⁶ PFU of wild-type virus ranged from1:625 to 1:3125. These data show that despite their attenuation forreplication and lethality in animals, the N-rearranged viruses eliciteda protective response that was undiminished compared with that of thewild-type virus.

Example 19 Organization of Genes to Develop an Optimum Vaccine Virus

The present invention illustrates that gene order in the Mononegaviralesdetermines the level of gene expression. Furthermore, these data showthat moving the important Nucleocapsid (N) gene away from its normal 3′promoter proximal position provides a means of generating sequentiallymore attenuated viruses. The maximal level of attenuation occurs whenthe N gene is placed next to last in the gene order. The highest levelof expression occurs from the 3′-most gene. Therefore, in constructing avaccine vector that is both attenuated and expresses high levels of theantigen involved in protection, the ideal arrangement is a combinationof N4 (3′-PMGNL-5′) or G1N2 (3′-GNPML-5′) or G1N4 (3′-GPMNL-5′). Inthese constructs, N4 is maximally attenuated and G1N2 yields thegreatest levels of the attachment glycoprotein, important for an immuneresponse. Based upon this criteria, G1N4 (3′-GPMNL-5′) should bemaximally attenuated and yield the highest levels of G protein.

Example 20 A Vaccine Vector Capable of Expressing Additional ForeignGenes so That the Level of the Foreign Gene is Regulated by Position

The genome of VSV can accommodate and express additional foreign genesif inserted at intergenic regions and if the conserved gene start, geneend and intergenic regions are maintained (FIG. 16) (Schnell et al.,1996). Additionally, the level of expression of a foreign gene insertedin the VSV genome can be controlled by the position in the genome atwhich the gene is inserted. A 660 nucleotide sequence of thebacteriophage Phi X174 genome surrounded by the conserved VSV gene startand gene end sequences was inserted into each sequential gene junctionof the full length cDNA of the VSV genome in such a manner so as tomaintain the conserved intergenic sequences. The gene order of theseconstructs was respectively: NIP (3′-NIPMGL-5′), PIM (3′-NPIMGL-5′), MIG(3′-NPMIGL-5′), or GIL (3′-NPMGIL-5′) where I represents the (I)nsertedforeign gene. Virus was recovered from each of the above-mentioned cDNAsby transfection as described above.

The viruses with the foreign gene sequence inserted at each position inthe genome were each used to infect BHK-21 cells and synthesis of RNAswas analyzed by metabolic labeling with [³H]-uridine in the presence ofactinomycin D. VSV genomic RNA and the 5 VSV specific mRNAs wereexpressed from all of the recovered viruses (FIG. 16). In addition, inall four cases, the synthesis of an mRNA of the size expected from theinserted foreign genetic material was also observed. The level ofexpression of the foreign gene varied as its position of insertion fromthe 3′ end of the genome. The highest level of expression was from NIP,followed by PIM, MIG and GIL (FIG. 16). Thus, these data show thatforeign genes may be inserted into the genome of VSV and that theforeign gene will be expressed if surrounded by the conserved VSV genestart and stop signals. Most importantly, this data shows that the levelof expression of the foreign gene is controlled by the position at whichthe gene is inserted into the genome.

Analysis of the growth potential of each of the viruses expressing aforeign gene showed that the position of the insertion of the foreigngene determined whether or not there was an effect on viral growth. NIPwas reduced by 10-fold in viral yields compared to wild-type virus,whereas PIM, MIG and GIL all replicated to levels equivalent to that ofwild-type virus. Thus, these data show that insertion of a foreign geneis possible, that it is not lethal to the virus, and that it may,depending on the position of insertion, serve to attenuate replication.

Example 21 Viruses and Cells

The San Juan isolate of the Indiana serotype of VSV provided thetemplate for all of the cDNA clone of the VSV genome except the Gprotein gene which was derived from the Orsay isolate of VSV-Indiana.All viruses were recovered from cDNAs in baby hamster kidney (BHK-21)cells. BHK-21 cells were also used for single-step growth assays andradioisotopic labeling of viral RNAs and proteins. Plaque assays wereperformed on the African green monkey cell line Vero-76.

Example 22 Plasmid Construction and Recovery of Infectious Virus

The construction of a full-length cDNA clone of the VSV genome and itsuse for the recovery of infectious virus has been described. Thisinfectious clone was manipulated using methods which allowed the genometo be assembled with the genes in different orders. No other changeswere made in the genome except for a single nucleotide in the intergenicregion downstream of the P gene. This change, from 3′-CA-5′ to 3′-GA-5′,has little effect on transcription.

To recover infectious viruses from the rearranged cDNA clones, BHK-21cells were infected with a recombinant vaccinia virus expressing the T7RNA polymerase (vTF7-3) (Fuerst, et al., 1986). One hour later the cellswere transfected with the rearranged VSV CDNA along with three plasmids,which expressed the N, P, and L proteins required for encapsidation andreplication of the anti-genomic RNA (Whelan et al., 1995). Infectiousviruses were harvested from the supernatant medium and amplified inBHK-21 cells at low multiplicity of infection (MOI) to avoid formationof DI particles and in the presence of cytosine arabinoside (25 μg/ml)to suppress the replication of vaccinia virus. Supernatant medium wasfiltered through 0.2 μM filters and the virus was banded on 15 to 45%sucrose velocity gradients to separate it from any remaining vTF7-3vaccinia virus. The gene orders of the recovered viruses were confirmedby amplifying the rearranged portions of the genomes using reversetranscription and PCR followed by restriction enzyme analysis.

Example 23 Analysis of Viral Protein Synthesis

Viral protein synthesis directed by each of the variant viruses wasmeasured in BHK-21 cells infected at a MOI of 50 with actinomycin D (5μg/ml) added at 3 hours post-infection. At 5 hours post-infection thecells were washed and incubated in methionine-free medium for 30 min.Cells were exposed to [³⁵S]methionine (30 μCi/ml, sp act 10.2 mCi/ml)for 1 hour. Cell monolayers were harvested directly into gel loadingbuffer and after normalizing for equal counts per minute (cpm) the viralproteins were separated on 10% polyacrylamide gels using a low bis toacrylamide ratio to separate the P and N proteins. Viral proteins werequantitated using a phosphorimager and the molar ratios calculated.

Example 24 Analysis of Virion Proteins

To assess the quantity of each of the proteins in the mature virions,BHK-21 cells were infected at a MOI of 5. After 2 hours the cells werewashed and incubated in methionine-free medium for 30 min. Cells werelabeled with [³⁵S]methionine (50 μCi/ml, sp act 10.2 mCi/ml) overnightwith cold methionine added to 10% of normal medium level. Supernatantfluid was collected, cell debris was removed by centrifugation, andvirus was collected by centrifugation through 10% sucrose. Afternormalizing the cpm, the viral pellet was resuspended in gel loadingbuffer and virion proteins separated on a 10% polyacrylamide gel. Virionproteins were quantitated using a phosphorimager and the molar ratiosdetermined.

Example 25 Single Cycle Virus Replication

BHK-21 cells were infected at a multiplicity of infection (MOI) of 3.After 1-hour adsorption the inoculum was removed and the monolayerwashed twice. Fresh medium was added and the cells incubated at 37° C.Supernatant fluids were harvested at indicated intervals over a 30-hourperiod and viral yields determined by plaque assay on Vero 76 cells.

Example 26 Lethality in Mice

Male Swiss-Webster mice, 3-4 weeks old, were purchased from TaconicFarms German-town, N.Y., and housed under BL2 containment conditions.Groups of 6 mice were lightly anesthetized with ketamine/xylazine andinoculated intranasally with 10 μl aliquots of serial ten-fold viraldilutions of the individual viruses in Dulbecco modified Eagle medium(DMEM). Control animals were given a similar volume of DMEM. Animalswere observed and each group was weighed daily. The 50% lethal dose(LD₅₀) for each of the viruses was calculated using the method of Reedand Muench (1938).

Example 27 Determination of Serum Antibody Levels and NeutralizationTiters

After virus inoculation blood was collected at weekly intervals fromgroups of 2-4 animals. Serum was pooled and heated to 57° C. for 40 minto inactivate complement. Cell monolayers infected with VSV wild-type(N1G4) and uninfected BHK-21 cells were lysed in detergent buffer (1%NP40, 0.4% sodium deoxycholate, 66 mM EDTA, 10 mM Tris-HCl pH 7.4) andused as antigen in a direct enzyme-linked-immunosorbant-assay (ELISA).Samples were serially diluted and detected using goat α-mouse Igconjugated to horseradish peroxidase. The optical density (OD) was readat 450 nm and the antibody titers calculated by linear regressionanalysis of a plot of optical density versus serum dilution. Theendpoint titers (log₁₀) were deduced at an OD 1.5 times the pre-immunesamples. Serum neutralizing antibody titers on day of challenge weredetermined by a standard plaque reduction assay on Vero 76 cells and thetiter expressed as the reciprocal of the dilution giving 50%neutralization.

Example 28 Protection of Mice From Wild-type Challenge

Mice were immunized intranasally with doses of each virus ranging from1-10,000 plaque-forming units (pfu) in DMEM. Twenty-one dayspost-inoculation groups of mice that received non-lethal doses of eachof the variant viruses were challenged intranasally with 5.4×10⁶ PFU ofN1G4 wild-type virus. Challenged animals and controls were monitored fora further twenty-one days. At weekly intervals blood was collected bytail bleeds for serum antibody titrations.

Example 29 Generation and Recovery of Rearranged Viruses

In the present work, cDNA clones were generated in which the G gene wasmoved from its normal position of fourth in the gene order, to thefirst, most promoter proximal position to increase its expression. Twonew gene rearrangements were generated: one in which the G gene wasmoved to first in the gene order and the remaining four genes were leftundisturbed to generate the order 3′-GNPML-5′ (G1N2), and the second inwhich the positions of the G and the N genes were exchanged to generatethe order 3′-GPMNL-5′ (G1N4), (FIG. 17). These cDNAs were transfectedinto cells and virus was recovered in both cases. The recovered viruseswere designated G1N2 and G1N4 respectively, according to the positionsof the N and G genes in the rearranged gene order. The properties ofthese viruses were examined in comparison to a virus derived from a cDNAclone created using the same gene rearrangement process to regeneratethe wild-type gene order (N1G4), and a virus with the gene order3′-P-M-G-N-L-5′ (G3N4).

Example 30 Effect of Gene Rearrangement on Viral Protein Expression

BHK-21 cells were infected with viruses with rearranged genomes and therelative levels of viral protein synthesis were examined by labeling for1 hr with [³⁵S]methionine at 5 hr post-infection. Total cellularproteins were resolved by SDS-PAGE and visualized by autoradiography. Atypical gel is shown in FIG. 18A. Infection with wild-type VSV and therearranged variants resulted in rapid inhibition of host proteinsynthesis which allowed the viral N, P, M, G, and L proteins to bedetected directly. Synthesis of G protein was significantly increasedrelative to the other viral proteins in cells infected with G1N2 andG1N4 viruses (FIGS. 18A and 18B, lanes 2 and 4) as compared to the ratein wild-type (N1G4) infected cells (FIGS. 18A and 18B, lane 1).

Proteins were quantitated by phosphorimaging. The molar percentage of Gprotein synthesized during a 1 hr labeling period was 2.3-fold higher inG1N2 infected cells and 1.7-fold higher in G1N4 infected cells than incells infected with wild-type virus. Similarly, translocation of the Ngene from its promoter proximal position to a more distal position inviruses G1N2, G3N4, and G1N4 decreased the rate of N protein synthesis(FIG. 18C). As a consequence of these changes in the relative rates ofsynthesis, the molar ratios of the viral proteins differed in cellsinfected with the variant viruses, in particular the ratio of N to Pwhich is known to be critical for optimal RNA replication (Pattnaik andWertz, 1990) (FIG. 18C).

The protein contents of purified virus particles were also examined todetermine if changes in protein synthesis in cells affected proteinassembly into virions. BHK-21 cells were infected with each of theviruses, labeled with [³⁵S]methionine overnight, and virions harvestedfrom supernatant fluids and separated from cell debris by centrifugationthrough 10% sucrose. Analysis of the virion proteins by SDS-PAGE (FIG.18B) showed no gross differences in the relative protein contents.Phosphorimager quantitation confirmed that despite the altered relativelevels of protein synthesis in infected cells the amounts of proteins invirions were similar to that of wild-type virus, with the exception ofvirus G1N2 in which the level of G was 1.6-fold higher than in wild-typeor the other rearranged viruses (FIG. 18D).

Example 31 Virus Replication in Cell Culture

Replication of the rearranged viruses under single-step growthconditions was examined in cultured BHK-21 cells infected at a MOI of 3followed by incubation at 37° C. Supernatant fluids were harvested atvarious times and the virus yields measured by plaque assay.Translocation of the N gene away from the promoter proximal positionresulted in stepwise reduction of replication as the gene was movedfurther from the first position. Movement of N to the second position(G1N2) decreased replication by 3-fold, whereas moving N to the fourthposition (G3N4) reduced replication by as much as 1,000-fold (FIG. 19).However the two viruses with N in the fourth position (G3N4 and G1N4)replicated to very different levels under single-step growth conditionspossibly because the molar ratio of N:P critical for optimal replicationwas less perturbed in G1N4 than G3N4. Measurement of the intracellularrates of protein synthesis 5 hours after infection showed a molar ratiofor N:P of 1:1.6 in cells infected with G1N4 (3′-GPMNL-5′) compared to aN:P ratio of 1:1.8 in G3N4 (3′-PMGNL-5′) infected cells (FIG. 18C). Amolar ratio for N:P of between 1:0.5 and 1:1 is optimal for replicationas shown by the N:P ratios of 1:0.7 in wild-type-infected cells (N1G4)and 1:0.8 for cells infected with G1N2. Both the wild-type virus andG1N2 have N directly followed by P in the gene order (FIG. 17). Too muchor too little P relative to N decreases replication significantly; thus,in cells infected with virus G3N4, not only is N limiting, but also themolar ratio of N:P is more than twice the optimal value. The kinetics ofreplication of G3N4 and G1N4 were delayed in comparison to wild-type andG1N2. Single-step growth of G3N4 and G1N4 was not complete until 24 hr.post-infection compared to 12 hr for N1G4 and G1N2. It is unlikely thatthe over abundance of G in the infected cell was responsible for thisdelay in replication since G1N2 showed no delay in replication relativeto wild-type virus.

Example 32 Lethality in Mice

Young mice provide a sensitive animal model for the study ofneuropathology caused by VSV and its mutants, (Sabin and Olitsky, 1938;Wagner, 1974) and inoculation of mice with wild-type VSV via theintranasal route results in fatal encephalitis. The pathogenesis of therearranged variant viruses was compared to that of wild-type virus afterintranasal inoculation in 3-4 week old Swiss-Webster mice. The dosesthat constitute an LD₅₀ for each of the viruses are shown in Table 4.

TABLE 4 LD₅₀ dose for mice of viruses with rearranged gene orders Viruspfu N1G4 (wt) 100 G1N2 50 G3N4 >100,000 G1N4 19,000 *The LD₅₀ valueswere calculated from the observed mortality among groups of 6 miceinoculated intranasally with a series of 10-fold dilutions of therearranged viruses. Virus titers were determined by plaque assay onVero-76 cells. Data from a single, internally controlled experiment isshown.

All the viruses were lethal for mice if given in sufficiently highdoses, although the doses of G3N4 administered in these experiments didnot reach the LD₅₀ seen previously. In general the position of the Ngene, the N:P ratio, and the resulting level of virus replication weremajor determinants of lethality. Viruses in which the N gene was movedaway from the promoter required greatly increased doses to constitute anLD₅₀. These results confirmed previous observations with viruses N1-N4in which the N gene was moved sequentially. However, the resultspresented here show that for viruses with N in the fourth position (G3N4and G1N4), both the replication ability and the LD₅₀ values wereaffected also by the position of the G gene.

The LD₅₀ values reported here are expressed in terms of the viral titerson Vero-76 cells which are about 10-fold higher than the titers onBSC-40 cells. Cell lines were changed because rearranging the gene orderof VSV could affect the interactions of the variant viruses with theinterferon system. BSC-40 cells are competent to produce interferonafter infection while Vero cells are not. Therefore changing to Verocells circumvented possible differences in interferon induction orsensitivity.

The first symptoms of sickness (a hunched posture and hind-limbparalysis) appeared 5 days post-inoculation with both N1G4 and G1N2viruses although the first deaths occurred earlier in animals inoculatedwith N1G4 (FIG. 20). The viruses with N in the fourth position inducedsymptoms more slowly and at a dose of 1,000 PFU per mouse, G3N4 inducedneither morbidity nor mortality, as observed before. In an attempt todetect sub-clinical signs of sickness the groups of mice were weigheddaily throughout the study period (FIG. 21). However, whereas the micethat showed symptoms invariably lost weight and died, those that showedno symptoms showed no weight differences from uninoculated controlanimals (FIG. 21). Similar results were observed after challenge of theinoculated mice with wild-type virus: all animals that developedsymptoms subsequently died and those that did not develop symptoms alsoshowed no weight loss.

Example 33 Serum Antibody

To assess the effect of inoculation of viruses with rearranged G geneson the humoral immune response, mice were inoculated intranasally with aserial 10-fold dilutions of each of the variant viruses. Blood wascollected at weekly intervals by tail bleed and the level of serumantibody determined by ELISA. Since survival of the inoculation was aprerequisite for this experiment, only doses at or below the LD₅₀ wereused. Translocation of the G gene changed the kinetics and magnitude ofthe antibody response (FIG. 22). Mice inoculated with wild-type virusmade barely detectable levels of antibody within 21 days, whereasanimals that received 100 PFU of G1N2 had significant titers by 14 daysand those given G1N4 had significant titers by 7 days post-inoculation.This accelerated and enhanced response can be seen most clearly bycomparing the mice that received 100 PFU (FIG. 22). The resultsdemonstrate that translocation of the G gene from the fourth to thefirst position enhances the humoral immune response to VSV. Mice givenG1N4 synthesized antibody earlier and at higher levels than those givenG3N4. This further confirms the observation that putting the G genefirst in the gene order increased the immunogenicity of VSV.

Twenty-one days post-inoculation, the mice were challenged with 5.4×10⁶PFU of wild-type VSV. A rapid increase in antibody titer was observed inanimals given either N1G4 or G1N2, although there was no further rise inthe already high titers that had been achieved prior to challenge inmice inoculated with G3N4 or G1N4.

Example 34 Neutralizing Antibody Titer After Inoculation

The level of neutralizing antibody in the serum at the time of challengewas measured. In mice and cattle, neutralizing antibodies are animportant element in protection against VSV infection. On the day ofchallenge mice were bled and serum samples were assayed for theirability to neutralize wild-type VSV in a standard plaque-reduction assayon Vero-76 cells. The reciprocal of the highest dilution that gave a 50%reduction of plaque numbers was calculated to determine the neutralizingtiters of the sera.

All the viruses with rearranged genomes elicited serum neutralizingantibody in mice (FIG. 23A). Neutralizing antibody was not detected atdoses of 1 or 10 pfu/mouse of either N1G4 or G1N2, but both viruseselicited detectable titers at doses of 100 pfu, the response to G1N2being 10-fold higher than that to wild-type virus. Thus for N1G4 andG1N2 the level of neutralizing antibody did not correlate with virusreplication in cell culture, where the wild-type virus replicated 2-3fold more abundantly than G1N2 (FIG. 19). This conclusion was reinforcedby the response to G3N4 and G1N4, which elicited approximately 10-foldhigher titers than the wild-type virus despite greatly reducedreplication potential.

In summary, viruses with over-expressed G and under-expressed N ininfected cells yielded increased levels of neutralizing antibodycompared to wild-type virus (N1G4) following intranasal inoculation. Thecombination of over-expressing G and under-expressing N combined thisenhanced immunogenicity with virus attenuation which allowed theadministration of higher doses that elicited correspondingly highertiters of neutralizing antibodies. Moreover, because of the lowerlethality of these viruses, 100 times more virus could be administeredwithout detriment, and under these conditions they elicited up to100-fold more neutralizing antibody than could be attained in responseto wild-type virus.

Example 35 Protection of Mice From Challenge

These results establish that non-pathogenic doses of the viruses thatover-expressed G protein could elicit significant humoral immuneresponses in mice. To see whether immunization with the rearrangedviruses could confer protection against VSV disease animals thatsurvived inoculated with each of the rearranged viruses were challengedafter 21 days with 5.4×10⁶ PFU of wild-type virus. This dose wassufficient to kill 83% of the uninoculated, age-matched, control groupof animals.

All the viruses with rearranged genomes conferred protection, the levelof which varied with the dose of inoculum (FIG. 23B). The levels ofprotection elicited by N1G4 and G1N2 were alike, reflecting thecomparable levels of replication and lethality of these virusesdescribed previously (FIG. 19 and Table 4). Similarly, the protectionconferred by G1N4 resembled that of G3N4. By 21 days post-inoculation,both viruses elicited solid immunity at doses of 1,000 PFU per mouse.Importantly, these fully protective doses were 20-100-fold less than thecorresponding LD₅₀ values. This emphasizes the conclusion that generearrangement is an effective method to systematically change thephenotype of VSV to optimize the properties required of a liveattenuated vaccine.

Discussion

The present invention demonstrates that the order of genes in negativestrand RNA viruses determines the level of gene expression. The geneorder can be rearranged and the levels of expression of the rearrangedviral genes reflects their position relative to the 3′ promoter oftranscription. By rearranging a single gene essential for replication,such as the N (nucleocapsid) gene, to successive positions down theviral genome, it is possible to affect the growth potential in cellculture and the lethality of the virus for mice in a stepwise manner.Thus, these data demonstrate a means of attenuating these viruses in astepwise manner. Attenuated viruses, such as N4 (3′-PMGNL-5′), are suchthat the lethal dose and the protective dose of the virus differ by over1000-fold, an attribute desirable for an attenuated vaccine candidate.

In addition, the present invention demonstrates that one may insertforeign genes into the genome of the negative strand virus, and recoverinfectious virus which expresses the foreign gene. The level ofexpression of the foreign gene can be controlled by the position in thegenome relative to the 3′ end at which the gene is inserted. The abilityof these viruses to accommodate foreign material is most likely due tothe fact that they possess helical ribonucleocapsids, such that thenucleocapsid and the virus both become larger as the size of the genomeis increased. No limit on the amount of foreign material that may beinserted has been reached.

The methodology of the present invention can be used to developattenuated viruses for vaccines, and such methodology is applicable toall members of the family Mononegavirales based upon the closesimilarity of the genome organization and mechanism for control of geneexpression for the members of the family. The Mononegavirales includethe Rhabdoviruses, such as rabies, the Paramyxoviruses, such as measles,parainfluenzaviruses, and respiratory syncytial virus, and theFiloviruses such as Ebola and Marburg.

The recovery of infectious viruses from cDNA clones of theMononegavirales permits experimental manipulation of the viral genome.Gene expression in these viruses is controlled at the transcriptionallevel by the order of the genes relative to the single promoter at the3′ end of the viral genome. A method to rearrange the order of the geneswithout introducing other changes into the genome was developed. Generearrangement altered the relative levels of synthesis of the viralproteins as expected, and produced infectious viruses having a varietyof different phenotypes. The present studies examined the consequencesof moving the G protein gene, which encodes the major neutralizingepitopes of the virus, from its promoter-distal position to first in thegene order. Expression of G protein in infected cells was significantlyincreased when its gene was moved from the fourth to the first position.However, the protein content of the purified virus particles was largelyunaffected by changes in the viral gene order.

The over-expression of G protein by these viruses allowed examination ofwhether they elicited an altered humoral immune response in animals. Thedata in FIG. 22 show that at an inoculum dose of 100 pfu, antibody wasproduced more quickly and at higher levels in animals infected with theviruses with G moved to a promoter proximal position as compared to thewild-type virus. Doses higher than 100 PFU could not be assayed for theN1G4 wild-type and G1N2 viruses because of their lethality. Whencompared at the dose of 100 pfu, viruses G1N2, G3N4, and G1N4 allelicited higher antibody titers more rapidly than wild-type virus. Thereduced lethality of the G1N4 and G3N4 viruses allowed higher doses tobe administered and in these cases antibody levels increased morerapidly than at lower doses.

The observation that all three viruses which had G move to a promoterproximal position elicited an enhanced humoral immune response in micehas implications for the understanding of protective immunity in thissystem. Although the relative levels of replication of the variantinocula in the cells that are most relevant for induction of the immuneresponse are unknown, it seems likely that they mirror, at leastqualitatively, the relative levels of replication seen in cell culture.If this is the case, G1N2, G3N4, and G1N4 express higher levels of Gprotein per inoculated mouse only during the first round of replication.After that, the more robust replication of the wild-type virus shouldhave more than compensated for its weaker G protein synthesis. Yet atthe same inoculated dose of 100 PFU per mouse, the variant viruseselicited an enhanced and accelerated humoral immune response compared tothe wild-type inoculated animals. It is remarkable that a modestincrease in the rate of G protein synthesis in infected cells shouldexert such a marked effect on the immune response, even in the face ofsubstantial attenuation of viral replication.

These results suggest that the kinetics and magnitude of the humoralimmune response becomes established very early in infection. Eitherthere is a short temporal window during which the scale of the immuneresponse becomes established irrevocably, or the immune response to VSVinfection is somehow determined by the level of G protein synthesis perinfected cell rather than by the aggregate immunogenic load. A similarconclusion is suggested by the efficacy of vaccines using recombinantcanarypox vectors under conditions where they are unable to replicate.Robust synthesis of antigen by a highly attenuated vector appears to bean effective vaccine strategy that warrants further exploration.

The position of the N gene and the level of N protein expressioncorrelated with efficiency of replication as the N protein is requiredin stoichiometric amounts for genomic RNA replication. The wild-typevirus N1G4 replicated to the highest titers, followed by virus G1N2 andviruses G1N4 and G3N4, which replicated least well. Virus G1N4 however,replicated significantly better than virus G3N4 although they both havethe N gene in the fourth position. Both of these viruses showed delayedreplication kinetics as might be expected if the formation of progenyvirus was limited by the supply of N protein.

It is known that the relative levels of the N and P proteins, inaddition to the absolute amount of N protein, are critical for efficientreplication. One function of the P protein is to maintain the N proteinin a soluble state such that it is able to support encapsidation ofnewly replicated RNA. Consistent with this, virus G1N2, while havingreduced N protein expression (FIGS. 18A and 18C) has the N and P genesin the same relative order as the wild-type virus N1G4 (FIG. 17).Accordingly, G1N2 expressed the N and P proteins at about the samerelative rates as wild-type virus, 1:0.8 and 1:0.7 respectively. Inagreement with this, virus G1N2 replicated only slightly less than thewild-type virus. Further to this point, although viruses G1N4 and G3N4both have N in the fourth position, G1N4 replicates substantially betterthan G3N4 (FIG. 19). The ratio between the rates of synthesis of the Nand P proteins is disparate from the wild-type in both of these viruses.However, virus G3N4 which has P in the first position has an N to Pratio in infected cells of 1:1.8 whereas the N:P ratio in cells infectedwith G1N4, where P is in the second position, is 1:1.6, closer to thatof wild-type virus. There is also a difference between these two virusesin the rates of G protein expression and it is possible that theincreased levels of Gprotein provide an advantage for replication ofvirus G1N4.

The reduced lethality of the viruses with gene rearrangements is alsoconsistent with the showing that attenuation of lethality in micecorrelated with reduced replication capacity. Reduced replication, inturn, was related to the overall expression levels of N protein and theN to P ratios as discussed above. Obviously any gene rearrangement whichbrings the G gene to the first position will displace the N gene fromits wild-type position and therefore decrease N protein expression. Itwill also alter the molar ratios of proteins whose gene positionsrelative to one another are changed by the rearrangement in question.Both types of change would be expected to alter replication efficiencyand lethality. The data in Table 4 show that the viruses which replicatebest, wild-type and G1N2, required only 50 to 100 PFU to constitute anLD₅₀ dose, whereas 200 to 1,000 times more G1N4 and G3N4 virus,respectively, were required for a lethal dose.

The data presented here show that rearrangement of genes allowed themanipulation of two important aspects of the viral phenotype: lethalityand the stimulation of neutralizing antibody. By reducing N proteinexpression, and altering the N:P ratio, it was possible to decreasereplication potential and lethality for animals; by increasing G proteinexpression it was possible to alter the kinetics and level of antibodysynthesis.

These results demonstrate that gene rearrangement can be used togenerate viruses with novel, beneficial phenotypes. This approachprovides the ability to alter the phenotype in a stepwise manner toachieve a desired level of attenuation or to alter the expression of aparticular gene. It allows the level of attenuation and immunogenicityto be modulated independently and systematically, exactly what is neededto generate and manipulate live attenuated vaccine candidates. Thisapproach should be applicable to other members of the Mononegavirales,all of which have a common mechanism for the control of gene expressionvia obligatorily sequential transcription originating from a single 3′promoter. Furthermore, viruses of the Mononegavirales have not beenfound to undergo homologous recombination, therefore changes made to thegene order should be irreversible by natural processes. Several foreigngenes have been expressed from VSV and in one study mice were protectedagainst the corresponding pathogen. These properties of VSV make it anexcellent candidate in which to generate future vaccines directedagainst VSV itself or against other pathogens. Studies designed toevaluate the pathogenesis and immunogenicity of the G1N2, G3N4, and G1N4viruses in a natural host are underway.

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules,and/or specific compounds described herein are presently representativeof preferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

12 1 38 DNA Artificial sequence PCR primer used to construct individualcDNA clones of VSV genes 1 acctgcacta acagaaaaaa actaacagag atgcaggt 382 23 DNA Artificial sequence starting plasmid to reconstruct therearranged full-length clones of N gene, containing a bacteriophage T7promoter followed by the VSV leader sequence 2 gaaactttaa cagtaatgca ggt23 3 22 DNA Artificial sequence starting plasmid to reconstruct therearranged full-length clones of L gene, containing the first 420nucleotides of the L gene 3 acctgcacta acagcaatca tg 22 4 14 DNAArtificial sequence primer_bind 1, 2, 3, 4, 5, 6, 7,8 n = a or g or c ort 4 nnnnnnnngc aggt 14 5 11 DNA Artificial sequence primer_bind 1, 2, 3,4, 5 n = a or g or c or t 5 nnnnngagac c 11 6 30 DNA Artificial sequenceprimer_bind 24, 25, 28, 29, 30 n = a or g or c or t 6 gggaagcttacctgcactaa cagnnatnnn 30 7 25 DNA Artificial sequence primer_bind 19,20, 23, 24, 25 n = a or g or c or t 7 tatgaaaaaa actaacagnn atnnn 25 834 DNA Artificial sequence primer_bind 16, 17 n = a or g or c or t 8ctttttttga ttgtcnntac gtccagggcc cacg 34 9 34 DNA Artificial sequenceprimer_bind 9 gcacccggga cctgcatatc tgttactttt tttc 34 10 34 DNAArtificial sequence primer_bind 10 gcacccggga cctgcatctc tgttagtttt tttc34 11 34 DNA Artificial sequence primer_bind 11 gcacccggga cctgcattgctgttagtttt tttc 34 12 34 DNA Artificial sequence primer_bind 12gcacccggga cctgcatatc tgttagtttt tttc 34

What is claimed is:
 1. A method of attenuating a virus of the orderMononegavirales, comprising the step of: rearranging the gene order ofsaid virus by moving a gene away from its wild-type position, whereinsaid gene is not the nucleocapsid gene of said viris.
 2. A method ofattenuating a virus of the order Mononegavirales, comprising the stepof: rearranging gene order of said virus by moving said virus'nucleocapsid (N) gene away from its wild-type 3′ promoter proximalposition and placing said N gene in the second or third position of saidgene order.
 3. A virus attenuated according to the method of claim 2.